A biosensor for quantifying an analyte in a sample and usage thereof are provided. The biosensor includes an insulative substrate, a cover, a sample supply port, an electrode system including at least a working electrode, a counter electrode and a third electrode, and a reaction layer at least formed over the working electrode. The third electrode, used for estimating whether the amount of the sample is sufficient, is disposed nearer to the sample supply port than the counter electrode is. Whether the amount of the added sample is sufficient is estimated by comparing the electrical current value detected between the working electrode and the counter electrode with the electrical current value detected between the working electrode and the third electrode. This invention has a simple structure and produces accurate measurements.